Acylated secreted proteins play essential roles in intercellular and organismal signaling pathways implicated in multiple diseases including diabetes and cancer. Protein-modifying members of the membrane-bound O- acyltransferase (MBOAT) enzyme family constitute key molecular control points for signaling through their roles in modifying the secreted proteins ghrelin, Hedgehog, and Wnt. Of these, ghrelin is unique in that it regulates feeding behavior and energy homeostasis. Since ghrelin requires acylation by GOAT for biological activity, a detailed understanding of this MBOAT family member is imperative to understand the role of ghrelin in disease and to target ghrelin-dependent pathways. However, the dearth of information regarding the structure, substrate binding sites, and catalytic mechanism of GOAT impedes understanding ghrelin signaling and development of small molecule tools to study the role of GOAT in metabolism-related diseases. There exists an urgent need to define the structural and catalytic foundations of protein acylation by GOAT, Hhat, and PORCN in order to understand the molecular basis of their diverse biological roles. The objective in this application is to define the structural and chemical basis for transmembrane protein acylation by a membrane O-acyltransferase. The studies proposed herein will develop a molecular-level structural model of GOAT verified and supported by chemical and biochemical studies. Supported by strong preliminary studies, our objective will be pursued in the following three Specific Aims: 1) Define the acyl donor and ghrelin binding sites within human GOAT (hGOAT); 2) Determine the hGOAT catalytic mechanism, and 3) Identify inhibitor binding sites within hGOAT. In the first Aim, an hGOAT structural model generated by bioinformatic analysis coupled with computational modeling will guide studies to identify the acyl donor and ghrelin binding sites within hGOAT, with the ultimate goal of defining how hGOAT accomplishes the topologically challenging transmembrane octanoylation of ghrelin. In the second Aim, structure-guided mutagenesis and mechanistic probes will reveal the location and composition of the active site responsible for ghrelin acylation. In the third Aim, inhibitor binding sites within hGOAT will be identified using cysteine-reactive chemical probes and computational docking studies. This proposal is innovative because it represents a new and substantive departure from the standard approaches for investigating the structure and mechanism of membrane-bound enzymes. Our work will establish a novel powerful and general approach for investigating structurally intractable membrane proteins. The proposed research is significant because it will generate the first structure of an MBOAT enzyme family protein acyltransferase while providing insight into the structures and catalytic strategies of MBOAT family members, which will advance the exploitation of acylated secreted proteins as therapeutic targets for human diseases.